Civilization has long recognized the relationship between fecal contamination and the outbreak of disease. Two categories of bacteria routinely tested as indicative of fecal contamination are Escherichia coli and total coliforms (typically Klebsiella sp., Citrobacter sp., and Enterobacter sp.). Because of the widespread testing for the presence of these two categories of microbial pathogens, substantial effort has been devoted to developing detection systems which permit quick and accurate testing of one or both of these bacterial groups.
Early methods for the detection of coliforms in food and water generally included the steps of (i) inoculating a primary culture medium--formulated to encourage growth of target pathogen(s) while discouraging the growth of others--with a specimen of the food/water to be tested, (ii) incubating the inoculated medium under conditions which promote growth of the target pathogen(s), and then (iii) analyzing the incubated medium to identify and/or quantify the microbes grown on the medium. The specimen introduced into the culture medium may be an untreated sample or a sample which has been treated to concentrate any target microbial pathogens present in the sample--such as by membrane filtration.
The three most common test methods for detecting coliforms are the Multiple Tube Fermentation technique (MTF), the Membrane Filtration technique (MF) and the Presence-Absence technique (P/A). The MTF technique yields the Most Probable Number (MPN) while the P/A technique provides a simple positive/negative answer. In contrast, the MF technique permits direct observation and counting of bacterial colonies. The MFT technique yields results in about twenty four hours (except for the determination of fecal coliforms which typically requires seventy two hours) but is difficult to interpret as sample turbidity increases and is typically less precise than MF data.
Because of the difficulty in effectively controlling the growth of other microbial pathogens in culture media, it is frequently necessary to isolate a sample of the suspected target microbial pathogen from the incubated primary culture medium, inoculate a second culture medium with the sample, incubate the second culture medium in order to obtain a purified sample of the microbe, and then test the purified sample to ensure that the suspected target pathogen is--indeed--the target pathogen. The need to conduct a second incubation greatly increases both the time required to conduct the test and the cost of the test.
Various media have recently been developed which include a detection system permitting differentiation of the target bacterial pathogen--such as Escherichia coli and/or total coliforms--from other bacteria in the primary medium. Such detection systems include a mechanism by which the target bacterial pathogen(s) is differentiated from other bacteria so as to prevent "false positive readings" while promoting sufficient growth and recovery of the target bacterial pathogen(s) so as to avoid "false negative readings". While simple in theory, formulation of culture media having such dual function detection systems has proven difficult in practice.
The industry has developed several acceptable test methods for detecting Escherichia coli and/or total coliforms but the search continues for superior culture media which can provide quick, accurate and easily readable results.
Examples of the various culture media which have been developed for detecting coliform bacteria and/or Escherichia coli are disclosed in International Application WO 89/04372 issued to Berg, European Patent Application Publication 0025467 issued to Rembach, U.S. Pat. No. 4,925,789 issued to Edberg, U.S. Pat. No. 5,210,022 issued to Roth et al., and the journal article New Medium for the Simultaneous Detection of Total Coliforms and Escherichia coli in Water by Brenner et al. published in Applied and Environmental Microbiology.
Berg (International Application WO 89/04372) discloses a culture medium formulated to quickly detect the presence/absence of coliforms. The medium includes lactose as a carbon nutrient, a methylumbilliferone substrate as a fluorescent indicator, and the anionic surface active agent sodium lauryl sulfate as a means of accelerating hydrolysis of the methylumbilliferone by the coliforms present in the medium.
A commercially available culture medium--sold under the trademark COLIFAST.TM.--is manufactured in accordance with the general precept disclosed in the Berg International Application. Briefly, COLIFAST.TM. mandates a maximum eight hour incubation period and is capable of detecting either fecal coliforms or total coliforms but not both on the same plate.
Rembach (European Patent Application Publication 0025467) discloses a culture medium formulated to detect the presence/absence of Escherichia coli which includes the chromogenic substrate 8-hydroxyquinoline glucuronide in combination with X-glucuronide as an activator. Rembach indicates that the medium results in the selective formation of a black precipitate in colonies of Escherichia coli.
Edberg (U.S. Pat. No. 4,925,789) discloses a culture medium formulated to detect the presence/absence of a target microbe by limiting the nutrients present in the medium to a nutrient which metabolizes to a unique detectible product (nutrient-indicator) and is capable of being detectably metabolized only by the target microbe. Edberg specifically discloses detection of Escherichia coli using a color-indicating nutrient selected from o-nitrophenyl-.beta.-D-glucuronide (yellow), p-nitrophenyl-.beta.-D-glucopyranosiduronic acid (yellow), .beta.-napthalamide-.beta.-D-glucuronide (purple), .alpha.-naphthol-.beta.-D-glucuronide (red) or methylumbilliferyl-.beta.-D-glucuronide (fluorescent) as the primary nutrient in the medium. Edberg further suggests that the medium may be used to simultaneously test for Escherichia coli and total coliforms (EC/TC) by employing different color-indicating nutrients for each of Escherichia coli and total coliforms in the medium.
A commercially available culture medium--sold by Environetics under the trademark COLILERT.TM.--is manufactured in accordance with the general precept disclosed in the Edburg '789 Patent. Briefly, the COLILERT.TM. culture medium detects the presence/absence of Escherichia coli and total coliforms (EC/TC) by limiting the nutrients present in the medium to o-nitrophenyl-.beta.-D-galactopyranoside (yellow) which is metabolized by total coliforms and methylumbilliferyl-.beta.-D-glucuronide (fluorescent) which is metabolized to a significant extent only by Escherichia coli.
Roth et al. (U.S. Pat. No. 5,210,022) discloses a culture medium formulated to quantify the amount of both Escherichia coli and total coliforms in a test sample. The medium limits the primary carbon nutrient in the medium to a chromogenic .beta.-galactosidase substrate producing an insoluble precipitate of a first color when reacted upon by .beta.-galactosidase (produced by coliforms generally) and a chromogenic .beta.-glucuronidase substrate producing an insoluble precipitate of a contrasting color when reacted upon by .beta.-glucuronidase (produced to a significant extent only by Escherichia coli). Roth et al. discloses that since the chromogenic indicators are specific to coliforms, the media need not include any inhibitors. While generally effective as an EC/TC testing medium, the medium of Roth requires an incubation period of about 24 to 48 hours and is difficult to read.
A commercial product designed to detect the presence of both Escherichia coli and total coliforms in a test sample is sold by RCR Scientific, Inc. under the trademark COLICHROME 2. The system is based upon the dual chromogenic and/or fluorescent system disclosed by Roth et al. and includes (i) a chromogenic .beta.-galactosidase substrate which produces a first color when hydrolyzed by .beta.-galactosidase [produced by coliforms generally], and (ii) a chromogenic .beta.-glucuronidase substrate (5-bromo-4-chloro-3-indoxyl-.beta.-D-glucuronide acid cyclohexylammonium salt) which produces a second color when reacted upon by .beta.-glucuronidase [produced to a significant extent only by Escherichia coli]. While generally effective as an EC/TC testing medium, the medium suffers from the same limitations as the medium disclosed by Roth et al.
Brenner et al., New Medium for the Simultaneous Detection of Total Coliforms and Escherichia coli in Water, Applied and Environmental Microbiology, 59:3535-3544 discloses a culture medium similar to that disclosed in Roth et al for quantify the amount of both Escherichia coli and total coliforms in a test sample. The medium includes a first chromogenic substrate which produces a fluorescent detectable color when reacted upon by .beta.-galactosidase (produced by coliforms generally) and a second chromogenic substrate producing a second color when reacted upon by .beta.-glucuronidase (produced to a significant extent only by Escherichia coli). While generally effective as an EC/TC testing medium, the medium is difficult to read and includes anionic detergents which tend to arrest recovery of stressed coliforms.
As evidenced by the abbreviated discussion set forth herein, several acceptable EC/TC detection systems are available. However, a substantial need continues to exist for a quick, accurate and easily readable culture medium which effectively encourages the growth of total coliforms while inhibiting the growth of non-coliforms and provides an observable differentiation between Escherichia coli and other coliforms within the medium.